Cystic fibrosis (CF) is the most common genetic disease of Caucasians in North America, occurring at a frequency of approximately 1 in 2500 births (Welsh et al., 1995). The disease results from defective function of the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein in a variety of tissues, including the pancreas and the lung epithelium. Riordan et al. (1989), Rommens et al. (1989) and Kerem et al. (1989) describe the cloning and sequencing of the CFTR gene. U.S. Pat. No. 5,543,399 to Riordan et al. discloses the purification of CFTR protein.
Normal CFTR protein is a membrane protein that functions as a cAMP-regulated chloride channel. The ΔF508 mutation in the CFTR gene, which is characterized by a deletion of the phenylalanine amino acid at position 508 of the CFTR protein, is the defect associated with most cases of CF. A CFTR protein having the ΔF508 mutation does not exit the ER and proceed on to the plasma membrane (Cheng et al., 1990; Gregory et al., 1991). It has been found that the ΔF508 mutation causes the temperature-sensitive misprocessing of the mutant protein that prevents the protein from exiting the ER (Denning et al., 1992).
The absence of CFTR protein in the pancreatic duct results in the blockage of the duct by a thick mucus that prevents pancreatic enzymes from passing from the pancreas to the intestine. Without treatment, CF patients decline as a consequence of malnutrition associated with insufficient pancreatic function. However, pancreatic enzymes may be introduced into the diet of CF patients as a means of reversing the effects of pancreatic insufficiency.
Unlike in the pancreas, the absence of CFTR function in lung epithelium results in a severe lung disease that cannot be readily reversed or controlled by conventional treatment. Lack of CFTR function in the lung results in airway fluid with an altered ion composition, thereby creating a favorable environment for disease-causing bacteria to colonize the lung. Additionally, mucus secreted into the lung becomes thick and viscous, preventing normal clearing of the bacteria from the airways. The chronic bacterial infection leads to destruction of lung tissue and loss of lung function. Current treatments for CF lung disease involve physical therapy to aid mucus clearance and antibiotic therapy to treat the lung infection. Although these treatments slow the progression of disease, they do not reverse it. Patients with CF consequently die prematurely, usually by the age of 30.
CF cells lack CFTR chloride channel activity because they have mutant CFTR genes that encode a defective CFTR protein. Thus, providing a patient with a copy of a normal human CFTR gene by way of gene therapy methods may provide an alternative to conventional therapies for the treatment of CF. Gene therapy strategies for the treatment of CF thus involve delivery of a normal wildtype human CFTR gene to mutant CF epithelial cells within the lung to restore normal CFTR chloride channel activity. Gene transfer of the CFTR gene can be accomplished by several different delivery methods. Recombinant viral vectors containing the wildtype CFTR gene provide one potential means to deliver the CFTR gene to CF cells. For example, recombinant adenovirus containing the wildtype CFTR gene have been shown to efficiently transfer the wildtype CFTR gene into CF epithelium, and correct the chloride channel defect (Welsh et al., 1994; Zabner et al., 1993). However, high doses of virus are generally required to obtain an efficacious response, which in time can cause inflammation resulting from the immune response to the viral proteins. Other viruses that might be used for CF gene therapy include AAV (Adeno-associated virus) (Flotte et al., 1994), retrovirus and lentivirus. The use of these viruses for gene therapy is also limited by the immune response to the high titer doses required for an efficacious response.
Gene transfer can also be achieved by transfection of CF cells by lipid-DNA complexes composed of plasmid DNA containing the CFTR cDNA in association with cationic or neutral lipids (Zabner et al., 1997). Gene therapy utilizing lipid-DNA complexes is a potential alternative to the use of viral vectors and presents a lower risk for an associated inflammatory immune response. However, gene transfer with lipid-DNA complexes is inefficient, so that only a small fraction of cells receive the therapeutic gene. As a consequence, only a very limited correction of the chloride channel defect is possible.
Another alternative for CF therapy is to identify drugs that have efficacy in treating the disease. However, the process of identifying potential drugs typically involves the screening of large numbers of compounds from a chemical library. Thus, the assay used to screen the library for active compounds must be specific for a desired activity, as well as rapid and cost effective. However, current drug screening strategies using mammalian cells and assays for CFTR chloride conductance are costly and labor intensive. Thus, there remains a need in the art for a means for rapidly screening potential drugs for the treatment of CF from among the hundreds of thousands of chemicals that can be tested.